In the recent past, light microscopic methods have been developed with which, based on a sequential, stochastic localization of individual point objects, in particular fluorescence molecules, image structures can be imaged that are smaller than the diffraction-dependent resolution limit of conventional light microscopes. Such methods are, for example, described in WO 2006/127692 A2; DE 10 2006 021 317 B3; WO 2007/128434 A1, US 2009/0134342 A1; DE 10 2008 024 568 A1; “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)”, Nature Methods 3, 793-796 (2006), M. J. Rust, M. Bates, X. Zhuang; “Resolution of Lambda/10 in fluorescence microscopy using fast single molecule photo-switching”, Geisler C. et al, Appl. Phys. A, 88, 223-226 (2007). This new branch of microscopy is also referred to as localization microscopy. The applied methods are known in the literature, for example, under the designations (F) PALM ((Fluorescence) Photoactivation Localization Microscopy), PALMIRA (PALM with Independently Running Acquisition), GSD (IM) (Ground State Depletion (Individual Molecule return) Microscopy) or (F) STORM ((Fluorescence) Stochastic Optical Reconstruction Microscopy).
The new methods have in common that the structures to be imaged are prepared with markers that have two distinguishable states, namely a “bright” state and a “dark” state. When, for example, fluorescent dyes are used as markers, then the bright state is a state in which they are able to fluoresce and the dark state is a state in which they are not able to fluoresce. For imaging image structures with a resolution that is higher than the conventional resolution limit of the imaging optical system, a small subset of the markers is repeatedly brought into the bright state and thus it is so to speak activated. In this connection, the activated subset is to be chosen such that the average distance of adjacent markers in the bright state is greater than the resolution limit of the imaging optical system. The luminance signals of the activated subset are imaged onto a spatially resolving light detector, e.g. a CCD camera. Thus, of each marker a light spot is detected whose size is determined by the resolution limit of the imaging optical system.
In this way, a plurality of raw data single frames is captured, in each of which a different activated subset is imaged. Using an image analysis process, then in each raw data single frame the centroids of the light spots are determined which represent those markers that are in the bright state. Thereafter, the centroids of the light spots determined from the raw data single frames are combined to a total representation. The high-resolution image created from this total representation reflects the distribution of the markers. For a representative reproduction of the structure to be imaged sufficient signals have to be detected. Since however the number of markers in the respective activated subset is limited by the minimum average distance which two markers may have in the bright state, a great many raw data single frames have to be captured to completely image the structure. Typically, the number of raw data single frames is in a range between 10,000 and 100,000.
The time required for capturing one raw data single frame has a lower limit that is predetermined by the maximum image capturing rate of the imaging detector. This results in relatively long total capturing times for a series of raw data single frames required for the total representation. Thus, the total capturing time can take up to several hours.
Over this long total capturing time, a movement of the specimen to be imaged relative to the imaging optical system may occur. Since for creating a high-resolution total image all raw data single frames are combined after the determination of the centroids, each relative movement between specimen and imaging optical system that occurs during the capturing of two successive raw data singles frames impairs the spatial resolution of the total image. In many cases, this relative movement results from a systematic mechanical movement of the system, also referred to as mechanical drift, which movement is caused, for example, by thermal expansion or shrinkage, by mechanical strains or by the change in the consistency of lubricants used in the mechanical components.
In the above-described high-resolution methods, it is of particular importance to ensure a drift-free positioning of the objective forming the imaging system relative to the specimen arranged on the platform. This can be achieved in that the objective is not, as usual, mounted to an objective revolver but directly to the platform. With such a design, the objective is arranged on the underside of the platform facing away from the specimen in the area of a through hole formed in the platform and images the specimen which is arranged on a specimen holder resting on the upper side of the platform through the through hole. As a result of the direct mounting of the objective to the platform, the path over which the objective is mechanically coupled to the specimen holder is relatively short, whereby a mechanical drift occurring between the objective and the specimen holder can largely be prevented.
When the objective is directly mounted to the microscope stage, then, however, the alignment of the objective to the optical axis of the microscope stand is relatively complex. For instance, the microscope stage has to be re-adjusted relative to the microscope stand whenever it has been removed from the microscope stand for example for repair purposes. This is laborious and error-prone.
From U.S. Pat. No. 3,572,889 A, a mechanism is known which serves to quickly mount different microscope stages to a microscope stand in an adjusted manner. This mechanism comprises an adjusting unit formed of a mounting bracket and a mounting plate arranged at the rear side of the mounting bracket. The mounting plate is fixed to a sliding element which is arranged vertically movably on the microscope stand. The microscope stage is coupled to the mounting bracket. The mounting plate and the mounting bracket have contact surfaces which allow a displacement of the mounting plate relative to the mounting bracket.
In US 2005/0083569 A1, a microscope is described having a focusing column, a stage and a mechanism which enables the removal of the focusing column from the stage. This mechanism comprises a threaded shaft mounted to the focusing column and a knurled wheel which is provided with a matching thread and can be rotated within the stage to couple and decouple the focusing column to and from the stage.